This invention relates to methods of amplifying target nucleic acids and, particularly, to methods of performing ligase chain reaction amplifications wherein at least one of the probes is reversibly modified at the ligation site so that it is not a substrate for the enzyme catalyzed ligation. Exemplary modifications include chemical blockage of reactant groups, or an abasic site and the addition of one or more nucleic acid bases to form an "overhang". The modified end prevents or reduces target independent spurious signal development and is later corrected in a target dependent manner to enable amplification.
Oftentimes, the feasibility of a nucleic acid based diagnostic assay is dependent on the ability to amplify the signal generated by only a few molecules of target. Although signal amplification is one potential solution, target amplification is often the preferred solution in nucleic acid based assays. Target amplification involves the repeated copying or duplication of sections of the nucleic acid designated as the target sequence.
One mechanism for target amplification is known as ligase chain reaction (LCRT.TM.). In LCR.TM., two primary probes (first and second, both of same sense) and two secondary probes (third and fourth, both of opposite sense with respect to primary probes) are employed in excess. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the 3' hydroxyl end of an "upstream" probe abuts the 5' phosphate end of a "downstream" probe, and so that a ligase can covalently ligate the two probes into a fused ligation product.
In like manner, LCR.TM. employs upstream and downstream secondary probes. A third probe (downstream secondary) can hybridize to the first probe (upstream primary) and a fourth probe (upstream secondary) can hybridize to the second probe (downstream primary) in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes can also hybridize to the target complement in the first instance. Once the fused strand of primary probes is separated from the target strand, it will hybridize with the third and fourth (secondary) probes which can be ligated to form a complementary, secondary fused product. In order to understand LCR.TM. and the improvements described herein, it is important to realize that the fused products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described more completely in EP-A-320 308, the entire disclosure of which is incorporated herein by reference.
One of the great strengths of amplification reactions is their ability to detect exceedingly small numbers of target molecules. However, it is important that the amplification process be highly specific since the amplification of non-target sequences along with signal could potentially impair the reliability of the amplification process. One potential problem associated with ligase chain reaction is background signal caused by target independent ligation of the probes. Since the third probe hybridizes to the first probe and the fourth probe hybridizes to the second probe, the probes, which are added in excess, can easily form duplexes among themselves. These duplexes can become ligated independently of the presence of target to form a fused product which is then indistinguishable from the desired amplified target, yet which is still capable of supporting further amplification. Although target independent ligation of these duplexes is a relatively rare event, it is sufficiently common to cause undesirably high background signals in highly amplified diagnostic assays.
EP-A-439 182 (corresponding to parent application Ser. No. 07/634,77 1 ) describes several mechanisms by which this background or spurious signal in LCR.sup.m can be reduced. One such mechanism involves 3' blocking groups or abasic sites that are "corrected" in the presence of target to yield ends that are ligation competent, i.e. ends that possess the 3' hydroxyl substrate necessary for ligation. The present invention expands and develops these mechanisms, particularly with regard to the use of endonuclease IV as the correction enzyme.
Levin, et al, "Metalloenzymes in DNA Repair", J. Biol. Chem. 266(34):22893-22898 (1991) have demonstrated that Endonuclease IV in native form contains zinc, and that inactive enzyme (purified in a metal free buffer) can be reactivated by the addition of certain divalent cations. In particular, Co.sup.2+ and Mn.sup.2 + at 200 .mu.M were effective to reactivate the enzymes, depending on the method (EDTA or 1,10-phenanthroline) of inactivation. Johnson and Demple, J. Biol. Chem. 263(34):18009-18016 (1988) have shown that the activity of a related enzyme, yeast 3' phosphoglycoaldehyde diesterase, is enhanced by concentrations of Co.sup.2+ from 3 .mu.M to about 3 mM, above which the cation was inhibitory.
A second potential problem associated with nucleic acid amplification systems is the potential for airborne and carryover contamination. Due to the exponential increase in target sequences, there is an increased potential for some of these molecules to contaminate an untested sample, and to render it falsely positive. Several methods have been described for reducing such contamination. They generally involve destroying substantially all the amplified products either immediately after amplification or immediately prior to the next amplification cycle.
One such contamination control method is taught in co-owned, co-pending application Ser. No. 07/863,622, filed Apr. 3, 1992.
Another method is taught by Walder, et al EP-A-496 483. This document describes the incorporation of ribonucleotides into PCR primers followed by destruction of the amplification products with RNase or alkaline hydrolysis. While the authors allege that their method is useful in transcription based amplification and in the ligase chain reaction, they have provided no conditions or demonstration of utility except in PCR.
It is well known in the art that DNA ligases will not ligate DNA probes hybridized to a fibonucleotide target. But WO91/17270 describes an LCR variation using fibonucleotide residues at the point of ligation. These residues can later be cleaved by alkali or enzymes to destroy the amplification product and prevent contamination.
However, there is no teaching of using fibo-modified probes in combination with 3' blocking groups as in the present invention. The present invention provides a mechanism for reducing or eliminating contamination in LCR.TM. using endonuclease IV correction methods. It has been discovered that DNA probes having a single ribonucleotide bearing a 3' blocking phosphate group can be used in LCR.TM.. When so used, the probes alleviate the background caused by target independent ligation and, at the same time, provide a mechanism to control contamination.